Identification and Separation of the Two Subunits of the Herpes Simplex Virus Ribonucleotide Reductase

ABSTRACT The present study reports the activity of BILD 1633 SE against acyclovir (ACV)-resistant herpes simplex virus (HSV) infections in athymic nude (nu/nu) mice. BILD 1633 SE is a novel peptidomimetic inhibitor of HSV ribonucleotide reductase (RR). In vitro, it is more potent than ACV against several strains of wild-type as well as ACV-resistant HSV mutants. Its in vivo activity was tested against cutaneous viral infections in athymic nude mice infected with the ACV-resistant isolates HSV type 1 (HSV-1) dlsptk and PAAr5, which contain mutations in the viral thymidine kinase gene and the polymerase gene, respectively. Following cutaneous infection of athymic nude mice, both HSV-1 dlsptk and PAAr5 induced significant, reproducible, and persistent cutaneous lesions that lasted for more than 2 weeks. A 10-day treatment regimen with ACV given topically four times a day as a 5% cream or orally at up to 5 mg/ml in drinking water was partially effective against HSV-1 PAAr5 infection with a reduction of the area under the concentration-time curve (AUC) of 34 to 48%. The effects of ACV against HSV-1 dlsptk infection were not significant when it was administered topically and were only marginal when it was given in drinking water. Treatment under identical conditions with 5% topical BILD 1633 SE significantly reduced the cutaneous lesions caused by both HSV-1 dlsptk and PAAr5 infections. The effect of BILD 1633 SE against HSV-1 PAAr5 infections was more prominent and was inoculum and dose dependent, with AUC reductions of 96 and 67% against infections with 106 and 107 PFU per inoculation site, respectively. BILD 1633 SE also significantly decreased the lesions caused by HSV-1dlsptk infection (28 to 51% AUC reduction). Combination therapy with topical BILD 1633 SE (5%) and ACV in drinking water (5 mg/ml) produced an antiviral effect against HSV-1 dlsptk and PAAr5 infections that was more than the sum of the effects of both drugs. This is the first report that a selective HSV RR subunit association inhibitor can be effective against ACV-resistant HSV infections in vivo.

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Escherichia coli and herpes-simplex-virus ribonucleotide reductase R2 subunit. Compared reactivities of the redox centres

Biochemical Journal ◽

10.1042/bj2900807 ◽

1993 ◽

Vol 290 (3) ◽

pp. 807-810 ◽

Cited By ~ 10

Author(s):

M Atta ◽

N Lamarche ◽

J P Battioni ◽

B Massie ◽

Y Langelier ◽

...

Keyword(s):

Escherichia Coli ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Ribonucleotide Reductase ◽

Active Sites ◽

Hydrophobic Interactions ◽

Chemical Reactivity ◽

Small Subunit ◽

Tyrosyl Radical ◽

Simplex Virus

Protein R2, the small subunit of ribonucleotide reductase, contains a diferric centre and a tyrosyl radical absolutely required for enzyme activity. The reduction of the tyrosyl radical and the mobilization of the iron centre result in the inhibition of the enzyme and thus of DNA synthesis. The chemical reactivity of the iron-radical centre of Escherichia coli and herpes simplex virus has been studied by u.v.-visible and e.p.r. spectroscopies. The tyrosyl radical is efficiently scavenged by hydroxamic acids and phenols during reactions controlled by steric hindrance and hydrophobic interactions. The reaction with o-disubstituted phenols yields the corresponding diphenoquinones. The reactivity of the bacterial radical greatly contrasts with that of the viral radical, and the iron centre in herpes-simplex-virus R2 is much more labile than that in E. coli R2, as shown from the facile mobilization of iron by chelators such as catechol. These results suggest that the active sites of the two enzymes are significantly different and might be useful for designing new antiviral agents.

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Study of ribonucleotide reductase in cells infected with six clinical isolates of herpes simplex virus type 2 (HSV-2) with mutations in its larger subunit

Virology ◽

10.1016/0042-6822(87)90193-0 ◽

1987 ◽

Vol 161 (1) ◽

pp. 249-251 ◽

Cited By ~ 3

Author(s):

Ashok R. Bapat ◽

Susan P. Grill ◽

Louise M. Nutter ◽

Yung-Chi Cheng

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Ribonucleotide Reductase ◽

Herpes Simplex Virus Type ◽

Clinical Isolates ◽

Simplex Virus ◽

In Cells

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Production of the R2 subunit of ribonucleotide reductase from herpes simplex virus with prokaryotic and eukaryotic expression systems: higher activity of R2 produced by eukaryotic cells related to higher iron-binding capacity

Biochemical Journal ◽

10.1042/bj3200129 ◽

1996 ◽

Vol 320 (1) ◽

pp. 129-135 ◽

Cited By ~ 14

Author(s):

Nathalie LAMARCHE ◽

Gilles MATTON ◽

Bernard MASSIE ◽

Marc FONTECAVE ◽

Mohamed ATTA ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Ribonucleotide Reductase ◽

Binding Capacity ◽

Eukaryotic Expression ◽

Eukaryotic Cells ◽

Expression Systems ◽

Simplex Virus ◽

R2 Subunit ◽

Eukaryotic Expression Systems

The R2 subunit of ribonucleotide reductase from herpes simplex virus type 2 was overproduced with prokaryotic and eukaryotic expression systems. The recombinant R2 purified by a two-step procedure exhibited a 3-fold higher activity when produced in eukaryotic cells. Precise quantification of the R2 concentration at each step of the purification indicated that the activity was not altered during the purification procedure. Moreover, we have observed that the level of R2 expression, in eukaryotic cells as well as in prokaryotic cells, did not influence R2 activity. Extensive characterization of the recombinant R2 purified from eukaryotic and prokaryotic expression systems has shown that both types of pure R2 preparations were similar in their 76 kDa dimer contents (more than 95%) and in their ability to bind the R1 subunit. However, we have found that the higher activity of R2 produced in eukaryotic cells is more probably related to a higher capability of binding the iron cofactor as well as a 3-fold greater ability to generate the tyrosyl free radical.

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